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Bacillus anthracis, the etiologic agent of anthrax, is genetically close to and commonly shares a giant gene pool with B. cereus and B. thuringiensis. In view of the human pathogenicity and the long persistence in the environment of B. anthracis, there is growing concern about the effects of genetic exchange with B. anthracis on public health. In this work, we demonstrate that an insecticidal plasmid, pHT73, from B. thuringiensis strain KT0 could be efficiently transferred into two attenuated B. anthracis strains, Ba63002R (pXO1+ pXO2−) and Ba63605R (pXO1− pXO2+), by conjugation in liquid medium in the laboratory, with transfer rates of 2.3 × 10−4 and 1.6 × 10−4 CFU/donor, respectively. The B. anthracis transconjugants containing both pHT73 and pXO1 or pXO2 could produce crystal protein Cry1Ac encoded by plasmid pHT73 and had high toxicity to Helicoverpa armigera larvae. Furthermore, the compatibility and stability of pHT73 with pXO1/pXO2 were demonstrated. The data are informative for further investigation of the safety of B. thuringiensis and closely related strains in food and in the environment.
In the Bacillus cereus sensu lato family, the classification of three species, B. thuringiensis, B. cereus, and B. anthracis, is based mainly on the presence of different functional plasmids in the clusters (26). B. thuringiensis is an insect pathogen, harboring plasmids encoding insecticidal proteins with toxicity against insects of the orders Diptera, Lepidoptera, and Coleoptera and nematodes (30); B. cereus is a ubiquitous food spoilage bacterium associated with two forms of human food poisoning (diarrheal and emetic syndromes), and the cereulide toxin (which causes the emetic syndrome) is encoded on a large virulence plasmid, pCERE01 (also named pCER270; ≈270 kb) (15); B. anthracis is the active agent of anthrax, and its virulence is attributed mainly to the presence of plasmids pXO1 (≈182 kbp) and pXO2 (≈95 kbp) since curing any of the plasmids attenuates the strain (5).
Recent studies demonstrated that the genetic backbones of both the pXO1 and pXO2 plasmids are not restricted only to B. anthracis but rather can be found in isolates of the related species B. cereus and B. thuringiensis as well (14, 24, 33). It was reported that B. cereus strain G9241, isolated from a patient with life-threatening pneumonia, carried a specific protective antigen gene (pagA) on plasmid pBCXO1 and a gene encoding a capsule made mainly of putative polysaccharide on plasmid pBC218 (14, 18). In further studies, the specific pXO1 fragments and pXO2 cap genes were detected in B. cereus strains 03BB102 and 03BB108 (13) and a backbone similar to the common backbone of pXO2 was found in a B. thuringiensis subsp. konkukian strain (serotype H34) isolated from a wounded soldier (12). These findings indicate that B. anthracis and related species may share a large gene pool and that genetic exchange among these species, followed by diversification, may be a major cause for their genomic plasticity and evolution.
The vegetative cells of the B. cereus sensu lato family strains can grow under nutrient-rich conditions as ubiquitous inhabitants, and they provide a large gene pool for horizontal gene exchange and coevolution, especially by conjugation. Some reports have confirmed that strains of B. thuringiensis and B. cereus exhibit low degrees of clonality and that exchange of genetic material between them occurs frequently in river water (31), soil (35), food preparation environments (33), and even the guts of insects (31, 37). Previous reports revealed that the toxin genes or whole toxin plasmids of B. anthracis can transfer to other Bacillus sp. strains in broth culture (28) and in the rhizosphere of grass plants (29) and that, conversely, exotic genes or plasmids can also be transferred into B. anthracis through conjugation. The frequent genetic change among B. cereus group strains raises concerns that B. anthracis and related species cannot be well distinguished by traditional species definitions and that the possible impact of B. cereus group strains on food and environments needs to be elucidated.
Our work focused on the conjugational transfer of the insecticidal plasmid pHT73 from B. thuringiensis into B. anthracis in liquid medium and in “ready-to-drink” milk. Our findings demonstrated that B. anthracis strains could receive the conjugative insecticidal plasmid pHT73 during food preparation and that pHT73 was stably coexistent with pXO1 or pXO2. Furthermore, the B. anthracis transconjugants could produce parasporal crystals with high toxicity to Helicoverpa armigera. The data are informative for further investigations into the safety of B. thuringiensis in food and the environment.
Strains and plasmids used in this study are listed in Table Table1.1. The erythromycin-resistant strain B. thuringiensis subsp. kurstaki KT0(pHT73-ErmR) (34) was used as the conjugational donor. The attenuated B. anthracis strains CMCC(B) 63002 and CMCC(B) 63605, carrying only plasmids pXO1 and pXO2, respectively, were used as recipients, and the recipient strains were designated Ba63002R and Ba63605R. Three strains of B. cereus were originally isolated from foodstuffs. Spontaneous mutants resistant to rifampin or streptomycin were selected in Luria-Bertani (LB) medium. pHT73-ErmR is a conjugative plasmid that carries a cry1Ac gene.
LB liquid medium and agar plates were used for strain culture and CFU counting (1). Ultrahigh-temperature (UHT)-sterilized whole milk was a commercial product (Wuhan, China) with a pH of 6.7. The sterility of the milk was tested by plating 100-μl samples onto LB agar plates and evaluating CFU after 48 h of incubation at 30°C. Antibiotics in agar plates and broth media were used at the following concentrations: erythromycin, 100 μg/ml; rifampin, 50 μg/ml; and streptomycin, 100 μg/ml.
The mating experiments were performed according to a modified procedure (2). The overnight precultures of donor and recipient strains were incubated separately with shaking (120 rpm) in either LB medium or milk (depending on the mating medium) without antibiotics for 10 h at 30°C. Equal amounts (500 μl) of donor and recipient strains were combined in 5 ml prewarmed mating medium and incubated at 30°C without shaking. After 5 h, 100-μl samples of the mating mixtures were subjected to a vortex for 10 s. Appropriate dilutions of mixtures were plated onto selective media for counting of donor, recipient, and transconjugant bacteria. In parallel, donor and recipient strains were grown separately under the same conditions as a control.
Five primer pairs were designed for detection of the specific genes in the donor and recipient strains and the resulting transconjugants. Primer pairs bcerm426S-bcerm929A and lep2A-lep2B were used for detecting the erythromycin resistance gene (16) and the cry1Ac gene (4), respectively, carried by pHT73; primer pair PA-1 (5′-TCCTAACACTAACGAAGTCG-3′) and PA-2 (5′-GAGGTAGAAGGATATACGGT-3′) was used for the pag gene carried by plasmid pXO1; primer pair Cap-1 (5′-ACTCGTTTTTAATCAGCCCG-3′) and Cap-2 (5′-GGTAACCCTTGTCTTTGAAT-3′) was used for pXO2 (the capsule gene cap); and primer pair Ba813-1 (5′-TTAATTCACTTGCAACTGATGGG-3′) and Ba813-2 (5′-AACGATAGCTCCTACATTTGGAG-3′) was used for a chromosomal marker, Ba813 (27). The random amplified polymorphic DNA (RAPD) analysis was performed with random primers OPA2 and OPA9 (Operon Technologies, Inc., Alameda, CA) to distinguish the donor from recipients and transconjugants (10).
Plasmid DNA was extracted from the donor, recipients, and transconjugants by the procedure described by Kado and Liu and was analyzed on 0.6% Tris-borate-EDTA agarose gel (19). The stability of the exterior plasmid in B. anthracis was investigated by a method described previously (16).
The donor, recipients, and transconjugants were incubated in LB medium until sporulation. Then the cultures were collected, washed with distilled water, and used for SDS-PAGE (20) and electron microscopy studies with a Hitachi H-7000FA microscope.
The toxicities of donor, recipient, and transconjugant strains against H. armigera were evaluated by bioassays (21). The target insects were a stable susceptible H. armigera colony maintained in the Experimental Insect Center, Wuhan Institute of Virology, Chinese Academy of Sciences. The bioassays of individual and combined sporulated cultures were performed with larvae confined singly to compartments of a 24-well tissue culture plate. The larvae were fed a semiartificial diet composed of wheat flour, soybean flour, yeast powder, and a mixture of vitamins and agar. Diluted recombinant B. thuringiensis sporulated cultures alone and in combination were well mixed with the melted feed at about 55°C, giving final concentrations ranging from 6.25 to 500 mg culture per g feed, and the mixtures were then transferred into separate cubes. One neonate larva was placed into each cell, and a plastic cover was used to confine the larvae. The plates were incubated at 26 ± 1°C, with humidity at 85% and a photoperiod of 12:12 h (light-dark). Duplicate sets of 24 larvae for each dose and five doses for each dose-response experiment were used. The bioassay was performed two or three times on different days, and the mortalities were recorded after 72 h. A 50% lethal concentration (LC50) with 95% fiducial limits was determined using probit analysis with a probit program (E. Frachon, Institut Pasteur), and the LC50 and LC90 were expressed in milligrams of culture per milliliter of artificial feed.
The mating experiment results indicated that attenuated B. anthracis strains Ba63002R and Ba63605R could receive the insecticidal plasmid pHT73 from the donor strain KT0 in LB broth medium and UHT-sterilized milk, with transfer ratios ranging from 6.9 × 10−4 to 1.9 × 10−7 transconjugants/donor (Table (Table2).2). It was evident that the conjugational transfer of the plasmid from B. thuringiensis to B. anthracis occurred with significantly higher frequency in UHT-sterilized milk (2.3 × 10−4 and 1.6 × 10−4 transconjugants/donor) than in LB medium (3.8 × 10−6 and 2.6 × 10−6 transconjugants/donor). All tested transconjugants retained the same phenotypes as the original recipient B. anthracis strains, being sensitive to penicillin G and unable to ferment salicin and having no hemolytic activity on blood agar (data not shown). The level of stability of plasmid pHT73 in the transconjugants was above 52% after culturing of the transconjugants for about 200 generations (Table (Table2).2). Two transconjugants, named KBa63002R and KBa63605R, were selected for further study.
Plasmid profiles indicated that the donor harbored a pHT73 plasmid of 75 kbp, that the recipients Ba63002R and Ba63605R contained a 185-kbp pXO1 plasmid and a 94-kbp pXO2 plasmid, respectively, and that the transconjugants harbored both their own anthrax plasmid and pHT73 (Fig. (Fig.1).1). The PCR analysis of specific marker genes confirmed that donor KT0 and transconjugants KBa63002R and KBa63605R contained the erythromycin resistance gene and the cry1Aa gene on plasmid pHT73, while the recipient strains Ba63002R and Ba63605R and transconjugants KBa63002R and KBa63605R contained the specific genes cap and pag and the marker Ba813 on plasmids pXO1 and pXO2, respectively (Fig. (Fig.2).2). Furthermore, the donor could be clearly distinguished from the recipients and transconjugants by RAPD analysis, all recipients and transconjugants exhibiting similar RAPD patterns (Fig. (Fig.33).
The donor and all transconjugants produced parasporal crystals during sporulation (Fig. 4B, D, E, and F), while no parasporal crystals could be observed in the two recipient strains (Fig. 4A and C). SDS-PAGE showed that strains that produced parasporal crystals gave a 130-kDa band of Cry1Ac (data not shown). Bioassay results indicated that the transconjugants derived from recipient B. anthracis strains had toxicity against larvae of H. armigera, with LC50s of 47.4 mg/ml (for KBa63605R) and 54.4 mg/ml (for KBa63002R), comparable with that of donor strain KT0 (Table (Table3).3). Contrarily, recipient B. anthracis strains exhibited no toxicity to the target insects at treatment concentrations as high as 500 mg culture per g feed.
Mating experiments demonstrated that plasmid pHT73 could be transferred from two transconjugants, KBa63002R and KBa63605R, to other B. cereus group strains and that all recipient stains (B. thuringiensis strain GBJ001 and B. cereus strains F3502/73S, MADM1279S, and AND1309S) could effectively receive the conjugative plasmid pHT73 with frequencies ranging from 9.4 × 10−4 to 9.1 × 10−6 transconjugants/donor (Table (Table44).
In this study, the conjugational transfer of an insecticidal plasmid, pHT73, from B. thuringiensis strain KT0 into attenuated B. anthracis strains in liquid media was evaluated. Our results demonstrated that the B. anthracis transconjugants harboring an insecticidal plasmid produced parasporal crystals during their sporulation and exhibited high toxicity against H. amergira insect larvae. This finding highlights the importance of gene exchanges among strains within the B. cereus group and the ecological impact of commercialized B. thuringiensis in the environment.
The results also showed that pHT73 can stably coexist with pXO1 or pXO2 in transconjugants. Previous studies revealed that pXO2 is a member of the pAMβ1 family of theta-replicating plasmids, which includes pXO2-like plasmids, pAW63 (originating from B. thuringiensis subsp. kurstaki), and pBT9727 (originating from B. thuringiensis subsp. konkukian) (23, 24, 32), and that the replicon of pHT73 belongs to the non-pAMβ1 family of theta-replicating plasmids (9, 36). Likewise, the replication gene repX and the quite recently recognized minimal replicon of pXO1 have no significant similarity to the replicon of pHT73 (23, 25). Thus, the three plasmids may utilize different replication systems, and there is no competition limiting their coexistence in a single cell.
There is no significant difference in the strain growth curve for bacteria cultured in LB medium and milk, while higher conjugational transfer frequencies were observed in the latter, which was consistent with the results of Modrie and colleagues (22). They observed earlier onset of conjugation of pAW63 in food matrices (e.g., milk and soya milk) than in LB medium, suggesting that the higher frequencies were caused by a higher transfer rate and/or longer mating period.
Although the insecticidal plasmid could be transferred into B. anthracis in liquid medium in the laboratory, no naturally occurring insecticidal B. anthracis strain has been reported until now. In natural environments, conjugational gene transfer from B. thuringiensis to B. anthracis rarely happens because B. anthracis exists mainly as dormant spores and the vegetative cells of B. anthracis survive poorly outside a host or complex artificial medium (6). However, plasmid transfer from other B. cereus group strains to B. anthracis, and vice versa, may occur under some specific conditions. It is well documented that the spores of insecticidal B. thuringiensis can germinate and then multiply in the midguts of dead target insects, and plasmid transfer at different frequencies under these conditions have been recorded previously (31, 34). The large-scale application of commercial B. thuringiensis for agricultural pest control may facilitate gene exchange among B. cereus group strains in target insects and then endow the strains with insecticidal properties and wider host ranges. However, the role of applied B. thuringiensis in horizontal gene transfer among B. cereus group strains needs to be further evaluated.
It is already known that members of the B. cereus group display high levels of chromosomal similarity and are phenotypically similar, and their species classification is based mainly on the presence of different functional plasmids (3). However, it has been noted previously that plasmids in B. thuringiensis and B. anthracis can be cured after continuous multiplication outside the target hosts (8), and also that the bacteria can acquire extra exotic plasmids through conjugational transfer in different environments. Our present results confirmed that the B. anthracis transconjugants harbored both native plasmid pXO1 or pXO2 and exotic pHT73, which have been reported to have different replication mechanisms (9). This finding makes the classification of separate species in this group more ambiguous. Therefore, further investigation of virulence genes related to anthrax in B. thuringiensis will be of importance for assessing its potential impact in food and in the environment.
We thank Didier Lereclus and Yang Rui Fu for kindly providing us with the B. thuringiensis subsp. kurstaki KT0 strain containing pHT73-ErmR and the DNA sample from Ba16.
The project was supported by grants (KSCX2-SW-301-10 and KSCX2-SW-315) from the Chinese Academy of Sciences, an NFSC grant (30470037), and a 973 grant (2009CB118902) from China.
Published ahead of print on 30 November 2009.